Peptide toxin formulation

ABSTRACT

Procedures are described which use solvents to increase the topical insecticidal activity of toxic insect peptides. These procedures comprise drying the peptides, if needed, followed by the addition of either: 1) a polar organic solvent, with or without water, to a dried peptide, or 2) the addition of polar aprotic solvent or other adjuvant to the dried peptide, followed by the addition of either: 1) a polar organic solvent, with or without water, (where a polar aprotic solvent is added first) or 2) a polar aprotic solvent or other adjuvant to the peptide polar organic solvent (where the polar organic solvent is added first), to the peptide formulation.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/528,402, filed Jun. 20, 2012, which is a divisional of U.S.application Ser. No. 12/568,400, filed Sep. 28, 2009, issued as U.S.Pat. No. 8,271,003, which claims the priority of U.S. ProvisionalApplication No. 61/101,825, filed Oct. 1, 2008, all of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the field of formulations for insecticidalpeptides.

BACKGROUND

Insecticidal peptides are toxic to their targets when deliveredinternally, but sometimes they have little or no topical activity.Topical insecticidal activity refers to a toxin's ability to inhibit thegrowth, impair the movement or even kill an insect when the toxin isdelivered to the insect or the insect's environment by spraying, orother means, as opposed to delivering the toxin directly to the insect'sgut or internal organs by injection or inducing the insect to consumethe toxin from its food, for example an insect feeding upon a transgenicplant.

The ability to successfully enhance or even change the properties ofpeptides with solvents has, until now, proven elusive. The wide variety,unique properties and special nature of peptides, combined with the hugevariety of possible solvents one could choose from, has produced only afew described methods for the enhancement of a few selected peptides inthe past 50 years or so. Various texts on the subject exist. See forexample, Principles of Dairy Chemistry Jenness and Patton (1959) pp.115-117, 127, 317, 326-328, 333.

Attempts have been made to enhance the activity of a few peptidesthrough purification and extraction. For example, U.S. Pat. No.5,840,838, Hensley, describes a procedure for enhancing the activity ofamyloid β peptide, a 39-43 residue peptide, with a process that involvesdissolving the peptide in an organic solvent, incubating it for 45minutes to 3 hours above room temperature, equilibrating to roomtemperature and then removing the solvent.

U.S. Pat. No. 4,530,784, Rosenberg, relates to a method of extracting abiologically active factor that restores contact inhibition of growth tomalignant cells in mammals by mixing specially prepared media with avolatile non-denaturing precipitating agent. The precipitate formed bythis reaction is separated from the formulation and extracted with abiologically acceptable ionic buffering agent.

U.S. Pat. No. 4,337,194, Diaz, is a process of preparing somatostatinusing a step-wise peptide coupling reaction in a solution of DMF. Theproduct of the reaction is isolated by evaporation or by precipitationwith a second solvent which renders the somatostatin insoluble, then thecrude peptide obtained is purified.

There are few if any descriptions, however, for a method to convert apeptide which has low topical insecticidal activity into one havingsignificantly greater topical insecticidal activity.

The procedure described here increases the topical insecticidal toxicityof insecticidal peptides. Peptides thus treated are referred to hereinas “enhanced topical peptides.” The process described herein of makingenhanced topical peptides is sometimes called making the peptides“special.” The process of making the peptides special makese thepeptides more active than before they are treated with the process ortreatment described herein. Once the peptides have been made specialthey can be applied topically to the insect, the insect's environment,to the places it inhabits, its habitat and to the food it touches, eatsor consumes; in order to control the insect, rather than having toengineer the peptide into the genome of a suitable plant or other food.Both the new process, the formulations, and the new enhanced topicalpeptides produced by the process are described and claimed herein.

SUMMARY OF THE INVENTION

Procedures are described which use solvents to increase the toxicity oftoxic insect peptides. Those procedures involve the preparation of thepeptides by drying the peptides, if needed, followed by the addition ofeither: 1) a polar organic solvent, with or without water, to a driedpeptide, or 2) a polar aprotic solvent or other adjuvant to the driedpeptide, followed by the addition of either: 1) a polar organic solvent,with or without water, (where a polar aprotic solvent is added first or2) a polar aprotic solvent or other adjuvant to the peptide polarorganic solvent (where the polar organic solvent is added first), to thepeptide formulation.

The procedures can also be described as follows: A method of increasingthe topical insecticidal activity of a toxic insect peptide, hereincalled making the peptide special comprising: adding either i) a polarorganic solvent or ii) a polar aprotic solvent or adjuvant to thepeptide and then adding either i) a polar organic solvent or ii) a polaraprotic solvent or adjuvant, which ever was not added initially to theinitial peptide formulation of above.

A method is described herein where the polar organic solvent comprisesfrom about 50, to about 99.9 percent (%) of the final volume of theformulation. The method is specifically described where the polarorganic solvent comprises from about 60, 70, 85, 90 to about 99.0percent (%) of the final volume of the formulation. The method isdescribed wherein the polar organic solvent comprises from about 70, toabout 99.0 percent (%) of the final volume of the formulation. Themethod is specifically described wherein the polar organic solventcomprises from about 60, 70, 80, 85, 90, to about 99.0 percent (%) ofthe final volume of the formulation. The polar organic solvent may beselected from acetone, methanol, ethanol, propanol and all its isomers,methyl ethyl ketone, diethyl ketone, acetonitrile, ethyl acetoacetate.The polar organic solvents selected from acetone, methanol, ethanol,propanol and all its isomers are especially useful.

The polar aprotic solvent or adjuvant will comprise from about 20%, toabout 0.001%, of the final volume of the formulation. Specifically thepolar aprotic solvent or adjuvant, comprises from about 15%, to about0.005%, from about 10%, to about 0.01%, from about 8%, to about 0.1%,from about 5%, to about 0.1%, of the final volume of the formulation.The polar aprotic solvent or adjuvant is selected from dimethylsulfoxide, dimethylformamide, dioxane and hexamethylphosphorotriamide.Dimethyl sulfoxide, also known as DMSO is exemplified.

The toxic insect peptides are preferably those with a) greater than 10amino acid residues and less than 3000 amino acid residues; b) amolecular weight from about 550 Da to about 350,000 Da; and c) they haveinsecticidal activity. The peptides may optionally have 1 to 5 disulfidebonds. The insecticidal activity of the peptides optionally are peptideshaving topical activity in at least one reproducable topicalinsecticidal assay. The toxic insect peptides may be selected from thevenom of a spider, mite, scorpion, snake, snails, certain plants or anycombination thereof. The spider may be an Australian funnel web spider,and peptides from the genus of Atrax or Hadronyche are easily madespecial using the procedures described herein. Specific peptides fromspiders, scorpions and plants are provided in the sequence listing.

Disclosed are formulations of special toxic peptides comprising: a) apeptide; b) a polar organic solvent; c) a polar aprotic solvent oradjuvant; d) wherein said polar organic solvent comprises from about 80,to about 99 percent (%) of the final volume of the formulation; e)wherein said polar aprotic solvent or adjuvant comprises from about 1,to about 10 percent (%) of the final volume of the suspension; and f) anoptional water phase, wherein said water phase comprises from 0 (zero),to about 10 percent (%) of the final volume of the suspension.

The peptides made special by the process of this invention are new andmay be separately claimed. These peptides are described by all of theirproperties and not simply their sequence. For the most part the peptidesequence information of the peptides which can be made special, asdescribed herein, are known; however, once treated the same peptideswill have greated topical activity. These peptides made special arenovel with unique properties, both the peptides and the process ofmaking them are disclosed and claimed herein.

Methods to control insects are also disclosed, in particularapplications of the special toxic peptides or special toxic peptideformulations applied to the insect's environment. The special toxicpeptide may be applied as a dry or liquid formulation. The formulationmay include wetting and dispersing agents, surfactants and other commoncomponents of insecticidal peptide formulations. Also, described arespecial toxic peptides produced as the product of any of the processesdescribed herein. The processes described herein can be used with anypeptides. The following peptides are mentioned by way of specificexamples and are not intended to limit the range, type or number ofpeptides can be made special using this process.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Active ingredient” means a peptide or polypeptide, herein it issometimes called a toxin.

“Insecticidal activity”, “insect control” or “control the insect” meansthat on or after exposure of the insect to the active ingredient, theinsect either dies, stops or slows its movement or its feeding, stops orslows its growth, fails to pupate, cannot reproduce or cannot producefertile offspring.

“Insect('s) environment” means any place or surface that is or will beexposed to an insect. The insect's environment includes the places itinhabits, its habitat and the food it touches, eats or consumes.

“Toxic insect peptide” means a peptide having insecticidal activity wheningested by or injected into an insect but having little, low or notopical insecticidal activity.

“Peptide made special” means the same as “Special topical peptide”below.

“Polar aprotic solvents” are organic solvents that have ion dissolvingpower but lack an acidic hydrogen. A polar aprotic solvent cannot donatehydrogen (H⁺ or proton.) These solvents generally have high dielectricconstants and high polarity. Further examples that should be consideredrepresentative and not limiting are; dimethyl sulfoxide (DMSO),dimethylftrmamide, dioxane, hexamethylphosphorotriamide and methylsulfoxide (MSO®). Adjuvants as polar aprotic solvents. Certain adjuvantscan also be polar aprotic solvents. Mixtures of oils with surfactants,commonly referred to as adjuvants are other examples of polar aproticsolvents. Crop oils in combination with surfactants can also act aspolar aprotic solvents. Examples such as Agicide Activator), Herbimax®,Maximizer®, and MSO® all available from Loveland company serve todemonstrate the commercial adjuvants can act as the polar aproticsolvents as the term is defined by this invention. MSO® is a methylatedseed oil and surfactant blend that uses methyl esters of soya oil inamounts of between about 80 and 85 percent petroleum oil with 15 to 20percent surfactant. Use and descriptions of MSO® used as a polar aproticsolvent can be found in Example 7.

“Polar organic solvents” are organic solvents with dipole momentssufficient to confer a dielectric constant of 15 or higher, acetonebeing one example. Other examples of polar organic solvents includecompounds with a dissociable H⁺, such as lower alkyl alcohols. Furtherexamples that should be considered representative and not limiting areas follows: acetone, methanol, ethanol, propanol, all isomers ofpropanol including 1- and 2-, propanol (n- and iso-propanol,respectively). Other polar organic solvents which might be successfullyused as part of this formulation can be determined by those skilled inthe art; these may include methyl ethyl ketone, diethyl ketone,acetonitrile, ethyl acetoacetate, etc.

“Special topical peptide” means a peptide previously having low topicalinsecticidal activity that has relatively higher topical insecticidalactivity because of the procedures described herein used to increase thetopical activity of peptides.

“Topical activity” or “topical insecticidal activity” means insecticidalactivity that results from exposure or contact of the insect's outerlayers, to the insecticidal peptide. Topical activity can result fromexposure to or contact between a treated material or surface and theexternal part of the insect, such as its feet, abdomen, antenna, mouth.Topical activity can result from insect preening of external parts ofthe insect followed by ingestion of the toxin. Treatment of any materialor surface with insecticide or toxin that then comes in contact with theinsect with resultant insecticidal activity is considered a topicalactivity.

“Topical application” means the application of the active ingredient toan insect's environment, or the insect itself. An insect's environmentmay be treated with a “topical application” in any manner including:spraying, painting, baiting, impregnating materials, such as treatingpaper or other objects that are then placed in the same area when theinsect is known or expected to visit or frequent. Topical applicationcan also mean direct contact with the insect and the insecticide.

The Process to Make Special

Description of the process to make peptides special.

Add a polar organic solvent, with or without water, to a dried peptideand then add a polar aprotic solvent or other adjuvant to the peptidepolar organic solvent (optional water) formulation, or in thealternative first add a polar aprotic solvent or other adjuvant to adried peptide and then add a polar organic solvent (with or withoutwater) to the polar aprotic solvent peptide formulation. Additionaltreatments and pretreatments to the peptides and peptide solventformulations are optional and are discussed below.

The peptides made special are then used as desired for effect.Application and use of the peptides made special may be with any means,either standard or as determined to be effective by a practicioner whois skilled in the art, including but not limited to: spraying through anatomizer or other type of spray nozzle, direct/indirect application ofdroplets of the formulation, application of the dried residue of theformulation to any body surface of the targeted insect, immersion of thetargeted insect in a bath, etc.

The peptide is made special upon completion of the addition of the polarorganic solvent and the polar aprotic solvent or other adjuvant, withthe solvents added in either order. It is better to start with a peptidethat is in a non-aqueous environment. The preferred order of solventaddition will be determined by a practitioner who is skilled in the artof insecticidal formulation, giving attention and consideration to theparticular peptides used. Numerous variations of the manner in which thesolvent is added can be made and should be apparent to one skilled inthe art. Some variations and more details of the procedure are providedbelow.

Preparation of the peptide by removing water may be needed if thestarting peptides are dissolved in water. The procedure of making thepeptides special may be practiced with peptides having either high orlow solubility in water. Often peptides are prepared in water basedsolvents or expressed in aqueous environments. If the peptide to be madespecial is in an aqueous environment, most of the water should first beremoved, i.e. the peptide should be dried. If the peptide is already ina dried state, then drying is not needed. Preparation of peptides caninvolve concentrating, purifying, isolating or identifying peptides andor the amounts or concentration of the peptide in the sample. Once thepeptide is in a preferred state, condition or concentration, it shouldbe “dried.” One method of drying a peptide, or taking it out of anaqueous environment, is to lyophilize the peptide using traditionalpeptide lyophilization procedures. See Protein Analysis and Purification2^(nd) Ed. Rosenberg 2005 pp 140. Other methods include, but are notlimited to, spray drying, rotary evaporation, and vacuum centrifugation.Peptide drying should be done in a manner such that the peptides are notunduly damaged or destroyed. Excessive heat should be avoided. Thoseskilled in the art will know and be able to practice appropriateprocedures to dry the peptide.

Adding the polar organic solvent to the dried peptide. Once the peptideis prepared by having most of the water removed, it is ready for mixingwith either the polar organic solvent or the polar aprotic solvent oradjuvant. Better results are sometimes obtained when the polar organicsolvent is added to the dried peptide before the polar aprotic solventis added and order is described below, but with some peptides in somesituations the polar aprotic solvent is added to the dried peptidefollowed by addition of the polar organic solvent.

The Polar Organic Solvent.

Many polar organic solvents can be used, some seem to produce peptideshaving greater topical activity than others. We have found the followingpolar organic solvents work well in this procedure to make peptidesspecial: acetone, methanol, ethanol, propanol, all isomers of propanolincluding 1- and 2-, propanol (n- and iso-propanol, respectively). Otherpolar organic solvents which might be successfully used as part of thisformulation can be determined by those skilled in the art; these mayinclude methyl ethyl ketone, diethyl ketone, acetonitrile, ethylacetoacetate, etc. The mixing of peptide and solvent can be done withany laboratory method of mixing such as vortex mixing, stirring,shaking, etc.

If the polar organic solvent is added to the dried peptide before theaprotic solvent/adjuvant then it (the polar organic solvent) can be asmuch as about 98 to 100% of the liquid in the formulation, and thepeptide will likely form a precipitate in the solvent. The formulationmay appear as a cloudy or hazy suspension. It should be vigorouslymixed. The polar organic solvent can have some water in it, see “Water”below, but pure or dry solvent also works well. Optimal finalconcentrations of water and polar organic solvent can be determined fora formulation of a particular special topical peptide by those skilledin the art. We have formulated special topical peptides with polarorganic solvent at final concentrations of 80 to 99%. Lowerconcentrations than 80% would also work with some peptides. Wespecifically describe polar organic solvents at final concentrations of60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98 and 99% and with water at final concentrations of 0% to 10%,in particular final water concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9and 10% are described. Those skilled in the art will be able tosuccessfully use values outside these sample ranges for particularformulations of particular special topical peptides.

Sonication. Once the peptide is properly taken up in the solvent it maybe sonicated or otherwise treated to further increase its topicalactivity. Sonication may break up the peptide particles in solution andreduce the size of the suspended particles. Sonication or otherprocedures to reduce particle size appears to increase topical toxicityof the peptides. Other procedures that reduce particle size, in additionto sonication, may be used to increase topical activity. Variousprocedures to reduce particle size should be known to those familiarwith peptide manipulation. Without being limited to any particularprocedure or mechanism, using a high speed blender, shaking or stirringwith glass beads may also be useful to increase the toxicity of thespecial toxic peptides.

Water. As mentioned above, the polar organic solvent does not need to bepure or absolute when used: it can contain water. Moreover, this watermay contain salts, organic molecules, peptides, etc. Water canpresumably be added to the peptide either before the polar organicsolvent is added to the peptide formulation, or at the same time orafter addition of the polar organic solvent. Care should be taken not touse too large a concentration of water, as we have observed that thiscan reduce or eliminate the activity of certain formulations of certainspecial topical peptides. The water need not be pure, it can includevarious proteins such as chitinases, phospholipases, lectins, etc., orsalts, sugars, carbohydrates, etc., in order to create a more useful andstable final solution. Alternatively, the water phase can initially bepure water and then various proteins such as chitinases, phospholipases,lectins etc. could be added to the water phase after it is mixed withthe polar organic solvent and the peptides.

The Polar Aprotic Solvent.

The polar aprotic solvent or adjuvant can be used as the firstingredient added to the dried peptide or it can be added to the peptidepolar organic solvent (water optional) formulation described above.Those skilled in the art can determine whether the polar aprotic solventor the polar organic solvent should be the first liquid added to thedried peptide in order to determine the better way to make the peptidesspecial. This determination will depend on the circumstances of eachcase and in particular exactly which toxic insect peptide is used andthe final formulation desired. However, such variations should bepracticed with care, as we have observed that in some cases lowerinsecticidal activity resulted if the polar aprotic solvent was added tothe peptide before the polar organic solvent is added.

Polar aprotic solvents lack an acidic hydrogen. These solvents generallyhave high dielectric constants and high polarity. Examples includedimethyl sulfoxide, dimethylformamide, dioxane andhexamethylphosphorotriamide.

The adjuvant can be any oil and/or emulsifying surfactant formulated foragricultural application of pesticides and especially peptides. Thesecommercial formulations typically have oils and emulsifying surfactantsformulated to “carry and spread” the active ingredients. Examplesinclude: “Aero Dyneamic” from Helena Chemical Co. which has methylatedor ethylated vegetable oil, a nonionic surfactant and a buffering agentor acidifier. It is further described as a “proprietary blend ofethoxylated alkyl phosphate esters, polyalkylene modifiedpolydimethylsiloxane, nonionic emulsifiers and methylated vegetableoils. For aerial use only at 2-8 qt/100 gal. 30-70 percent. Provides pHreduction and buffering, NIS and oil blend” See label for rates. Furtherexamples and manufactures of adjuvants can be found in Table 1.

TABLE 1 Agrochemical Surfactants. Suggested Surfactant/AdjuvantComposition Brief Description Application LI 700 Phosphatidylcholine,Non-ionic low 8-24 oz/100 gallon (Loveland methylacetic acid, andfoaming penetrant; (0.0625%-0.1825% Products) alkyl polyoxyethylene aidsin providing solution) ether (80%); Constituents uniform sprayineffective as spray coverage and to adjuvant (20%) acidify spraysolutions SILWET L-77 Polysiloxane polyether Non-ionic, 3-16 oz/100 gal(Loveland copolymer, polyether organofunctional (0.02%-0.125% Products)(100%) silicone surfactant solution) which lower's surface tension belowcommonly used surfactants, resulting in more effective wetting and moreuniform coverage MSO Concentrate Methylated vegetable oil, Enhancesactivity of 1-2 pints per acre w/LECI-TECH alcohol ethoxylate, postapplied (1.25-2.5% based on (Loveland phosphatidylcholine herbicidesnon-ionic 10 gal/acre) Products) (100%) surfactants and petroleum-basedcrop oils TACTIC Synthetic latex, 1,2- Increases adherence 8-32 oz/100gal. (Loveland propanediol, Alcohol (latex polymer) and (0.0625%-0.25%Products) ethoxylate, silicone coverage solution) polyether copolymer(organosilicone) (63.4%); Constituents ineffective as spray adjuvant(36.6%)

Optimal final concentrations of polar aprotic solvent and/or adjuvantcan be determined for the formulation of a particular topical peptidemade special by those skilled in the art. We have successfullyformulated special topical peptides with polar aprotic solvent at finalconcentrations of 10% and as low as 0.01%, with 0.5% working well. Theadjuvant Silwet L-77, for example, works well at a final concentrationas low as 0.01%, and those skilled in the art should be able tosuccessfully find other adjuvants using even higher or lower values thanthe ranges described here for particular formulations of particulartopical peptides made special.

The water and sonication steps described above can be applied in anyorder. Particular modes of insecticidal application for particularformulations of special topical peptides will be determined by thoseskilled in the art.

Topical Toxic Peptides and their Preparation.

Examples of toxic insect peptides are well known and can be found innumerous references. They can be identified by their peptidic nature andtheir activity, usually oral or injection insecticidal activity. Here weprovide a few examples to better illustrate and describe the invention,but the invention is not limited to these examples. All of theseexamples and others not shown here are descriptive of new materials,described and claimed here for the first time.

Toxic insect peptides are peptides of greater than 5 amino acid residuesand less than 3000 amino acid residues. They range in molecular weightfrom about 550 Da to about 350,000 Da. Toxic insect peptides have sometype of insecticidal activity. Typically they show activity wheninjected into insects but most do not have significant activity whenapplied to an insect topically. The insecticidal activity of toxicinsect peptides is measured in a variety of ways. Common methods ofmeasurement are widely known to those skilled in the art. Such methodsinclude, but are not limited to determination of median response doses(e.g., LD₅₀, PD₅₀, LC₅₀, ED₅₀) by fitting of dose-response plots basedon scoring various parameters such as: paralysis, mortality, failure togain weight, etc. Measurements can be made for cohorts of insectsexposed to various doses of the insecticidal formulation in question.Analysis of the data can be made by creating curves defined by probitanalysis and/or the Hill Equation, etc. In such cases, doses would beadministered by hypodermic injection, by hyperbaric infusion, bypresentation of the insecticidal formulation as part of a sample of foodor bait, etc.

Toxic insect peptides are defined here as all peptides shown to beinsecticidal upon delivery to insects either by hypodermic injection,hyperbaric infusion, or upon per os delivery to an insect (i.e., byingestion as part of a sample of food presented to the insect). Thisclass of peptides thus comprises, but is not limited to, many peptidesproduced naturally as components of the venoms of spiders, mites,scorpions, snakes, snails, etc. This class also comprises, but is notlimited to, various peptides produced by plants (e.g., various lectins,ribosome inactivating proteins, and cysteine proteases), and variouspeptides produced by entomopathogenic microbes (e.g. the Cryl/deltaendotoxin family of proteins produced by various Bacillus species.)

The following documents are incorporated by reference in the US in theirentirely, in other jurisdictions where allowed and they are of commonknowledge given their publication. In addition they are incorporated byreference and known specifically for their sequence listings to theextent they describe peptide sequences. See the following:

-   US patents: U.S. Pat. No. 5,763,568, issued Jun. 9, 1998,    specifically the sequences in the sequence listing, and those    numbered 1-26, and those known as “kappa” or “omega” toxins,    including those that can form 2-4 intrachain disulphide bridges, and    the peptides appearing on columns 2 and 4, and Table 5, and in FIG.    5, FIG. 15, FIG. 16, FIG. 17, FIG. 18. U.S. Pat. No. 5,959,182,    issued Sep. 28, 1999, specifically the sequences in the sequence    listing, and those numbered 1-26 and those known as “kappa” or    “omega” toxins, including toxins that can form 2-4 intrachain    disulphide bridges, and the peptides appearing on columns 2 and 4,    and Table 5, and in FIG. 5, FIG. 15, FIG. 16, FIG. 17, FIG. 18. U.S.    Pat. No. 6,583,264 B2, issued Jun. 24, 2003, and U.S. Pat. No.    7,173,106 B2, issued Feb. 6, 2007 specifically sequence number 1,    named “omega-atracotoxin-Hv2a or ω-atracotoxin-Hv2a, including    toxins that can form 2-4 intrachain disulphide bridges. U.S. Pat.    No. 7,279,547 B2, issued Oct. 9, 2007, specifically the sequences in    the sequence listing, and those numbered 1-35, and variants of    o-atracotoxin-Hv2a, toxins that can form 2-4 intrachain disulphide    bridges, and the peptides appearing on columns 4-8 of the    specification, and in FIG. 3 and FIG. 4. U.S. Pat. No. 7,354,993 B2,    issued Apr. 8, 2008 specifically the peptide sequences listed in the    sequence listing, and those numbered 1-39, and those named U-ACTX    polypeptides, toxins that can form 2-4 intrachain disulphide    bridges, and variants thereof, and the peptides appearing on columns    4-9 of the specification and in FIG. 1. EP patent 1 812 464 B1,    published and granted Aug. 10, 2008 Bulletin 2008/41, specifically    the peptide sequences listed in the sequence listing, toxins that    can form 2-4 intrachain disulphide bridges, and those as numbered    1-39, and those named U-ACTX polypeptides, and variants thereof, and    the peptides appearing in paragraphs 0023 to 0055, and appearing in    FIG. 1.

Described and incorporated by reference to the peptides identifiedherein are homologous variants of sequences mentioned, have homology tosuch sequences or referred to herein which are also identified andclaimed as suitable for making special according to the processesdescribed herein including but not limited to all homologous sequencesincluding homologous sequences having at least any of the followingpercent identities to any of the sequences disclosed her or to anysequence incorporated by reference: 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or 95% or greater identity to any and allsequences identified in the patents noted above, and to any othersequence identified herein, including each and every sequence in thesequence listing of this application. When the term homologous orhomology is used herein with a number such as 30% or greater then whatis meant is percent identity or percent similarity between the twopeptides. When homologous or homology is used without a numeric percentthen it refers to two peptide sequences that are closely related in theevolutionary or developmental aspect in that they share common physicaland functional aspects like topical toxicity and similar size within100% greater length or 50% shorter length or peptide.

Described and incorporated by reference to the peptides identifiedherein that are derived from any source mentioned in the US and EPpatent documents referred to above, including but not limited to thefollowing: Toxins isolated from plants and insects, especially toxinsfrom spiders, scorpions and plants that prey on or defend themselvesfrom insects, such as, funnel web spiders and especially Australianfunnel web spiders, including toxins found in, isolated from or derivedfrom the genus Atrax or Hadronyche, including the genus species,Hladronyche versuta, or the Blue Mountain funnel web spider, Atraxrobustus, Atrax formidabilis, Atrax infensus including toxins known as“atracotoxins,” “co-atracotoxins,” “kappa” atracotoxins, “omega”atracotoxins also known as ω-atracotoxin, U-ACTX polypetides,U-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hvlb, or mutants or variants,especially peptides of any of these types and especially those less thanabout 200 amino acids but greater than about 10 amino acids, andespecially peptides less than about 150 amino acids but greater thanabout 20 amino acids, especially peptides less than about 100 aminoacids but greater than about 25 amino acids, especially peptides lessthan about 65 amino acids but greater than about 25 amino acids,especially peptides less than about 55 amino acids but greater thanabout 25 amino acids, especially peptides of about 37 or 39 or about 36to 42 amino acids, especially peptides with less than about 55 aminoacids but greater than about 25 amino acids, especially peptides withless than about 45 amino acids but greater than about 35 amino acids,especially peptides with less than about 115 amino acids but greaterthan about 75 amino acids, especially peptides with less than about 105amino acids but greater than about 85 amino acids, especially peptideswith less than about 100 amino acids but greater than about 90 aminoacids, including peptide toxins of any of the lengths mentioned herethat can form 2, 3 and or 4 or more intrachain disulphide bridges,including toxins that disrupt calcium channel currents, including toxinsthat disrupt potassium channel currents, especially insect calciumchannels or hybrids thereof, especially toxins or variants thereof ofany of these types, and any combination of any of the types of toxinsdescribed herein that have topical insecticidal activity, can be madespecial by the processes described herein.

Venomous peptides from the Australian Funnel Web Spider, genus Atrax andHadronyche are particularly suitable and work well when treated by themethods, procedures or processes described by this invention. Thesespider peptides, like many other toxic peptides, including especiallyare toxic scorpion and toxic plant peptides, become topically active ortoxic when treated by the processes described by this invention.Examples of suitable peptides tested and with data are provided herein.In addition to the organisms mentioned above, the following species arealso specifically know to carry toxins suitable for being made specialby the process of this invention. The following species are specificallynamed: Agelenopsis aperta, Androctonus ausrdalis Heclor,Antraxformidabillis, Anrax infensus, Alrax robustus, Bacillusthuringiensis, Bothus mnarlensii Karsch, Bothus occilanus tunetanus,Buthacus arenicola, Buthotus judaicus, Buthus occitams mardochei,Centruroides noxius, Ceniruroides suffusus suffusus, Hadronyche ifensa,Hadronyche versuta, Hadronyche versutus, Hololena curia, Hottertoilajulaica, Leiurus quinquesiriarus, Leiurus quinqueslriatus hebraeus,Leiurus quinquestriatus quinqueslriatus, Oldenlandia afinis, Scorpiomaurus palmatus, Tityus serrulatus, Titylu zulianu. Any peptidic toxinsfrom any of the genus listed above and or genus species are suitable forbeing made special according to the process in this invention.

The Examples in this specification are not intended to, and should notbe used to limit the invention, they are provided only to illustrate theinvention.

As noted above, many peptides are suitable candidates as the subject ofthe process to make special. The sequences noted above, below and in thesequence listing are especially suitable peptides that can be madespecial, and many of these have been made special according to thisinvention with the results shown in the examples below.

(one letter code) SEQ ID NO: 60SPTCI PSGQP CPYNE NCCSQ SCTFK ENENG NTVKR CD1   5    10    15    20    25    30    35 37 (three letter code)SEQ ID NO: 60Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys Pro Tyr Asn Glu Asn 1               5                   10                 15Cys Cys Ser Gln Ser Cys Thr Phe Lys Glu Asn Glu Asn Gly Asn Thr             20                  25                 30Val Lys Arg Cys Asp          35     37Named “ω-ACTX-Hv1a” it has disulfide bridges at positions: 4-18, 11-22and 17-36. The molecular weight is 4096.

(one letter code) SEQ ID NO: 117GSSPT CIPSG QPCPY NENCC SQSCT FKENE NGNTV KRCD1   5    10    15    20    25    30    35   39 (three letter code)SEQ ID NO: 117Gly Ser Ser Pro Thr Cys Ile Pro Ser Gly Gln Pro Cys Pro Tyr Asn 1               5                   10                  15Glu Asn Cys Cys Ser Gln Ser Cys Thr Phe Lys Glu Asn Glu Asn Gly             20                  25                  30Asn Thr Val Lys Arg Cys Asp          35              39Named “ω-ACTX-Hv1a+2” it has disulfide bridges at positions: 6-20, 13-24and 19-38. The molecular weight is 4199.

(one letter code) SEQ ID NO: 118GSAIC TGADR PCAAC SPCCP GTSCK AESNG VSYCR KDEP1   5    10    15    20    25    30    35   39 (three letter code)SEQ ID NO: 118Gly Ser Ala Ile Cys Thr Gly Ala Asp Arg Pro Cys Ala Ala Cys Cys 1               5                   10                  15Pro Cys Cys Pro Gly Thr Ser Cys Lys Ala Glu Ser Asn Gly Val Ser             20                  25                  30Tyr Cys Arg Lys Asp Glu Pro           35             39Named “rK-ACTX-Hv1c” it has disulfide bridges at positions: 5-19, 12-24,15-16, 18-34. The molecular weight is 3912.15

(one letter code) SEQ ID NO: 119GSQYC VPVDQ PCSLN TQPCC DDATC TQERN ENGHT VYYCR A1   5    10    15    20    25    30    35    40 41 (three letter code)SEQ ID NO: 119Gly Ser Gln Tyr Cys Val Pro Val Asp Gln Pro Cys Ser Leu Asn Thr 1               5                   10                  15Gln Pro Cys Cys Asp Asp Ala Thr Cys Thr Gln Glu Arg Asn Glu Asn             20                  25                  30Gly His Thr Val Tyr Tyr Cys Arg Ala          35                  40  41Named “rU-ACTX-Hv1a (“Hybrid”)+2” it has disulfide bridges at positions:5-20, 12-25, 19-39. The molecular weight is 4570.51

Preparation of the Topical Toxic Peptides

The toxic peptides described above can be prepared in a variety of waysand in some embodiments they need not be prepared by any formal process.The peptides can simply be collected with or without other impurities ina composition and utilized. In one embodiment in which several Examplesare provided below, the peptides are lyophylized or they have some, mostor nearly all liquid removed prior to being made special. In someembodiments the peptides are still wet and only excess liquid isremoved. In some embodiments the peptides are in aqueous solutions or insomething similar to an aqueous solution. The peptides need not beisolated or purified prior to being made special.

Reproducable Assays to Measure Topical Insecticidal Activity.

The topical insecticidal activity of a peptide can be measured andquantified. Numerous assays are available. Several examples ofreproducable assays useful to determine the topical activity of apeptide are provided in the examples below. These examples describe bothpeptide and assay in detail but they should not be used to limit thescope of the claims or invention.

Materials And Methods

EXAMPLES Example 1 Topical Assay with Acetone and DMSO using House FlyToxin is ω-ACTX-Hv1a:

(SEQ ID NO: 60) SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD

Synthetic. Molecular weight: 4050 Da. LD₅₀ in House-fly: 90.2 μmol/g

Administration and application of the formulation.

Insect is House fly (Musca domestica) from Benzon research weighingbetween 12-20 mg (average mass 16 mg) would each receive 2 μLmicropipette applications of formulations onto the dorsal thoracicsurface of the body.

Toxin Doses:

90,000 μmol/g w-ACTX (1000×Injection LD50) dissolved in 90% Acetone/10%DMSO or DMSO (10%-20%) with 0.1% Tween 20,

9,000 μmol/g (100×Injection LD50), and

900 μmol/g (100×LD50) dissolved in DMSO (10-20%) with 0.1% Tween 20.

Preparation of Water-Based Application Solutions

ω-ACTX/DMSO stock-3.5 mg lyophilized ω-ACTX (Auspep) massed anddissolved in 70 μL DMSO (50 μg/μL stock). Water+Tween stocks−1000 μLaliquots of Tween 20 stocks were prepared in water to the percent volumeto volume values (listed below) from a 1% Tween 20 stock (e.g. 111 μL 1%Tween 20+889 μL water for the 0.111% Tween 20 stock, etc.). In allcases, the Tween 20 stock was added to the tube first, then DMSO, andfinally the ω-ACTX/DMSO stock.

TABLE 2 Water-DMSO Treatments. ω-ACTX Dose [DMSO] Water + Tween Final(pmol/g) (%) ω-ACTX DMSO (% v/v) Volume 90,000 10% 17.5 μL ω-ACTX STOCK12.5 μL   270 μL 300 μL (50 μg/μL in DMSO) (0.111% Tween) −ve 10% — 30μL 270 μL 300 μL (0.111% Tween) 90,000 20% 17.5 μL ω-ACTX STOCK 42.5μL   240 μL 300 μL (50 μg/μL in DMSO) (0.125% Tween)  9,000 20% 30 μL90,000 pmol/g ACTX 54 μL 216 μL 300 μL Solution (0.138% Tween)   900 20%30 μL 9,000 pmol/g ACTX 54 μL 216 μL 300 μL Solution (0.138% Tween) −ve20% — 60 μL 240 μL 300 μL (0.125% Tween) Note. In Table 2 and manyTables below some or all of the following abbreviations are used:“twitch” or “twch” means twitching; “morb” means moribund and “−ve”means “negative control conditions” which is the same as experimentalconditions but without any active ingredient (s).

Preparation of Acetone-Based Application Solutions:

Acetone—The same 50 μg/μL ω-ACTX stock in DMSO was used to create aformulation in 90% acetone and 10% DMSO that would deliver a doseequivalent of 90,000 pmol/g when applied as a 2 μL droplet to tohouseflies of an average mass of 16 mg. In this case, the toxin stockwas added to the acetone first, and then a final volume of DMSO wasadded to reach 10% m/v DMSO. This was done to examine the amount ofprecipitate when dissolved toxin was added to acetone.

A second ω-ACTX solution was also prepared by dissolving 1.2 mglyophilized ω-ACTX (Auspep) in 240 μL acetone (5 μg/μL stock). 50 μL ofthis stock was diluted in 121.5 μL of Acetone after which 17.15 μL DMSOwas added (10% concentration). Calculations leading to an estimate theω-ACTX dosage for this formulation are as follows:

-   -   50 μL×5 μg/μL ω-ACTX stock=250 μg ω-ACTX+171.5 μL total        volume=1.458 μg/μL×2 μL/insect=2.915 μg/insect    -   2.915 μg/insect×1 μmol/4050 μg×10⁶ pmol/μmol=719.7 pmol/insect×1        insect/0.016 g=45,000 μmol/g

A control formulation of bovine serum albumin (BSA) was also prepared inacetone and DMSO. Due to the concentration of the stock BSA, theconcentration of acetone was only about 60% in 10% DMSO.

TABLE 3 Acetone-DMSO Treatments DMSO ω-ACTX Dose Concentration Final(pmol/g) (%) ω-ACTX DMSO Acetone Volume 90,000 10% 17.5 μL ω-ACTX 12.5μL     270 μL 300 μL STOCK (50 μg/μL in DMSO) 45,000 10% 50 μL (5 μg/μLin 17.15 μL   104.3 μL 171.5 μL   Acetone) −ve 10% — 30 μL   270 μL 300μL +ve 10% 87.5 μL 10 μg/mL BSA 30 μL 182.5 μL 300 μL

Administration and Application of the Formulation

Houseflies were refrigerated for ˜4 hr and then anesthetized with CO₂.Each treatment formulation described above was applied to a group oftenanesthetized flies. The treatments consisted of a 2 μL droplet of therespective formulation, pipetted onto the dorsal thoracic body surfaceof a fly. Groups often anesthetized flies were used to test eachtreatment regime. The Acetone/DMSO solution rapidly evaporated from thecuticle. The DMSO formulations were allowed to absorb through thecuticle. Treated flies which revived on their dorsal surface tended tostick to the bottom of the bin and struggle following placement withfood and water; intervention was made to prevent this by gently tappingthe bin or manipulating stuck flies back to an upright orientation withtweezers. A control group of untreated flies was also reserved to ensuremortality was not affected by CO₂ exposure. All treatments were givenfood and water and observed for 24 hours.

Results (n=10 for all treatment groups, number of dead flies per groupreported in second column):

TABLE 4 Results of Acetone-DMSO Treatments Dead (Time Treatment (TimeApplied) post treatment) Notes −ve 20% DMSO/Tween (3:54 PM 7/1/08) 0 (~8hr) All flies healthy/active 90,000 pmol/g ω-ACTX 20% DMSO/Tween 1 (~8hr) 1 dead in food, others (4:09 PM 7/1/08) healthy 9,000 pmol/g ω-ACTX20% DMSO/Tween (4:22 PM 1 (~7.5 hr) 1 stuck to bottom(?) 7/1/08) dead(?)900 pmol/g ω-ACTX 20% DMSO/Tween (4:32 PM 0 (~7.5 hr) 7/1/08) −ve 10%DMSO/Tween (5:39 PM 7/1/08) 0 (~6.5 hr) 1 stuck to bottom & dislodged90,000 pmol/g ω-ACTX 10% DMSO/Tween 0 (~7 hr) (4:52 PM 7/1/08) −ve 90%Acetone/10% DMSO (5:25 PM 7/1/08) 1 (~7.5 hr) +ve BSA Protein (5:01 PM7/1/08) 1(?) (~7 hr) 1 unresponsive on side of food dish 90,0000 pmol/gω-ACTX (DMSO Stock) 0 (~7 hr) 1 stuck on back & 90% Acetone/10% DMSO(5:11 PM 7/1/08) dislodged 45,0000 pmol/g ω-ACTX (Acetone Stock) 1 (~6.5hr) 1 dead in food dish 90% Acetone/10% DMSO (5:19 PM 7/1/08) −veuntreated (5:40 PM 7/1/08) 0 (~6.5 hr) −ve 20% DMSO/Tween (3:54 PM7/1/08) 0 (~19 hr) All flies healthy/active 90,000 pmol/g ω-ACTX 20%DMSO/Tween 1 (~19 hr) 1 twitching (4:09 PM 7/1/08) 9,000 pmol/g ω-ACTX20% DMSO/Tween (4:22 PM 1 (~18.5 hr) Dead from sticking to 7/1/08)bottom 900 pmol/g ω-ACTX 20% DMSO/Tween (4:32 PM 0 (~18.5 hr) All flieshealthy/active 7/1/08) −ve 10% DMSO/Tween (5:39 PM 7/1/08) 1 (~17.5 hr)Dead in food 90,000 pmol/g ω-ACTX 10% DMSO/Tween 1 (18 hr) 2 twitching(4:52 PM 7/1/08) −ve 90% Acetone/1.0% DMSO (5:25 PM 7/1/08) 1 (~17.5 hr)+ve BSA Protein (5:01 PM 7/1/08) 1 (~18 hr) 90,0000 pmol/g ω-ACTX (DMSOStock) 1 (~18 hr) 1 twitching 90% Acetone/10% DMSO (5:11 PM 7/1/08)45,0000 pmol/g ω-ACTX (Acetone Stock) 5 (~17.5 hr) 2 twitching 90%Acetone/10% DMSO (5:19 PM 7/1/08) −ve untreated (5:40 PM 7/1/08) 0(~17.5 hr) All flies healthy/active −ve 20% DMSO/Tween (3:54 PM 7/1/08)0 (~27.5 hr) 90,000 pmol/g ω-ACTX 20% DMSO/Tween 3 (~27.5 hr) 1twitching (4:09 PM 7/1/08) 9,000 pmol/g ω-ACTX 20% DMSO/Tween (4:22 PM 1(~27 hr) 7/1/08) 900 pmol/g ω-ACTX 20% DMSO/Tween (4:32 PM 0 (~27 hr)7/1/08) −ve 10% DMSO/Tween (5:39 PM 7/1/08) 1 (~26 hr) 90,000 pmol/gω-ACTX 10% DMSO/Tween 3 (~26.5 hr) 1 twitching (4:52 PM 7/1/08) −ve 90%Acetone/10% DMSO (5:25 PM 7/1/08) 1 (~26 hr) +ve BSA Protein (5:01 PM7/1/08) 1 (~26 hr) 90,0000 pmol/g ω-ACTX (DMSO Stock) 3 (~26.5 hr) 1twitching 90% Acetone/10% DMSO (5:11 PM 7/1/08) 45,0000 pmol/g ω-ACTX(Acetone Stock) 7 (~26 hr) 1 sick 90% Acetone/10% DMSO (5:19 PM 7/1/08)−ve untreated (5:40 PM 7/1/08) 0 (~26 hr)

Topical application of ω-ACTX dissolved in Acetone with 10% DMSO wasinsecticidal to flies (70% mortality at 24 hrs.) while a similarpreparation of ω-ACTX dissolved in DMSO then diluted to 10% DMSO inacetone was less insecticidal (˜30%). These results are similar to thetopical assays in which ω-ACTX dissolved in Acetone with DMSO added to aconcentration of 10% killed 90% of houseflies (Jun. 19, 2008) whileω-ACTX dissolved in DMSO and then diluted in Acetone to a concentrationof 90,000 μmol/g killed 40% of treated insects. Topical application ofω-ACTX in 10-20% DMSO in water was also insecticidal, but considerablyless so than the Acetone/DMSO solution (30% vs. 70%).

Example 2 Topical Assay with Acetone/Methanol/DMSO using House Fly

Toxin is ω-ACTX-Hv1a:

(SEQ. ID. NO. 60) SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCDSynthetic. Molecular weight: 4050 Da. LD₅₀ in House-fly: 90.2 pmol/g.

Administration and Application of the Formulation

Insects: House fly (Musca domestica) from Ienzon research weighingbetween 12-18 mg (average mass 15 mg); 2 μL micropipette applicationonto dorsal thorax.Cabbage Looper (Trichoplusia ni) from Benzon research weighing ˜30 mg; 2μL micropipette application to dorsal anterior.Toxin Dose Calculations: ˜90,000 μmol/g ω-ACTX (1000×Injection LD₅₀),0.015 g/fly×90,000 pmol/g=1350 μmol/fly×4050 pg/pmol×1 μg/10⁶ pg=5.467μg/insect.5.467 μg/2 μL application=2.733 μg/μL×150 μL=410.06 μg×1 μL/5 μg=82 μL 5μg/μL ω-ACTX stock

Preparation of Application Solutions.

Mixtures of ω-ACTX in acetone (90%) and DMSO (10%) were preparedaccording to Table 5 from a stock preparation of 1.5 mg lyophilizedω-ACTX dissolved in 300 μL of acetone to produce a 5 mg/mL solution.ω-ACTX formed a cloudy precipitate when acetone was added which settledout when left on the bench. The preparation was vortexed for ˜5 sec tohomogenize the precipitate prior to dilution.

Methanol/DMSO—Mixtures of ω-ACTX in methanol (90%) and DMSO (10%) wereprepared according to Table 5 from a stock preparation of 2.3 mglyophilized ω-ACTX dissolved in 460 μL of methanol to produce a mixturewith a final peptide concentration of 5 mg/mL. ω-ACTX formed a cloudyprecipitate when methanol was added which settled out when left on thebench, in a similar manner to atracotoxin/acetone suspensions. Thepreparation was vortexed for ˜5 sec. to homogenize the precipitate priorto dilution.

TABLE 5 Treatment Preparations. Total Treatment ω-ACTX DMSO SolventVolume 90,000 pmol/g 82 μL 5 mg/mL 15 μL  53 μL Acetone 150 μL ω-ACTXSTOCK in Acetone −ve — 15 μL 135 μL Acetone 150 μL 90,000 pmol/g 82 μL 5mg/mL 15 μL  53 μL Methanol 150 μL ω-ACTX STOCK in Methanol −ve — 15 μL135 μL Methanol 150 μL

Table 5 treatment preparations are are formulations ofacetone/methanol/DMSO; order of addition when preparing each formulationwas solvent, ω-ACTX stock (where necessary), and finally DMSO.

Administration and application of the formulation.Each treatment formulation described above was applied to a group oftenCO₂-anesthetized houseflies. Treatment application consisted of a 2 μLdroplet of the respective formulation, pipetted onto the dorsal thoracicbody surface of a fly. Each mixture was vortexed immediately prior toeach application to ensure suspension of precipitate particles.Following treatment, insects were placed in bins with fresh food andwater, allowed to recover, and observed over 24 hours.

Each treatment formulation described above was also applied to a groupoften 2^(nd) instar T. ni. Treatment application consisted of a 2 μLdroplet of the respective formulation, pipetted onto the anterior dorsalbody surface. Each mixture was vortexed and immediately prior to eachapplication to ensure suspension of precipitate particles. All treatmentmixtures were vortexed immediately prior application to ensuresuspension of precipitate particles. Following treatment, insects wereplaced on fresh media and observed for 24 hrs.

TABLE 6a Housefly Dead Dead Treatment (18 hrs.) (24 hrs.) 90,000 pmol/gAcetone + 4 4 DMSO −ve Acetone + DMSO 0 0 90,000 pmol/g Methanol + 10 10DMSO −ve Methanol + DMSO 0 0 Untreated 0 0

TABLE 6b Cabbage Looper Treatment Dead (18 hr) Dead (24 hr) 45,000pmol/g Acetone + 0 0 DMSO −ve Acetone + DMSO 0 0 45,000 pmol/gMethanol + 0 0 DMSO −ve Methanol + DMSO 0 0

Table 6a and Table 6b (above) Results of administration ofacetone/methanol/DMSO formulations to Housefly and Cabbage Looper.

Topical treatment of houseflies with a high dose of ω-ACTX in methanolwith DMSO was insecticidal with 100% mortality at 18 hours posttreatment compared to only 40% mortality of houseflies treated withω-ACTX in acetone. There was no control mortality in either treatment.There was no difference between ω-ACTX and control treatments of cabbageloopers in terms of insect death, feeding, or behavior. Methanolpotentiated the topical activity of w-ACTX more than acetone in thisexperiment.

Example 3 Topical Assay with Methanol and Ethanol using Housefly Toxinis ω-ACTX-Hv1a

(SEQ ID. NO: 60) SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCDSynthetic. Molecular weight: 4050 DaLD₅₀ in House-fly: 90.2 pmol/g

Administration and Application of the Formulation

Insect: House fly (Musca domestica) from Benzon research weighingbetween 12-20 mg (average mass 16 mg). 2 μL micropipette applicationonto dorsal thorax.

Toxin Dose Calculations:

90,000 μmol/g ω-ACTX (1000×Injection LDSo),

0.015 g/fly×90,000 pmol/g=1350 pmol/fly×4050 pg/pmol×1 μg/10⁶ pg=5.467μg/insect5.467 μg/2 μL application=2.733 μg/μL×250 μL=683.43 μg×1 μL/5 μg=136.7μL 5 μg/μL ω-ACTX stockNOTE: Treatment solutions calculated for flies of an average mass of 15mg (12-18 mg range); actual average mass of insects used was 16 mg(12-20 mg range). ω-ACTX dose in tables below has been adjusted for thisdiscrepancy.

Preparation of Application Solutions

Methanol—Solutions of ω-ACTX in Methanol were prepared according toTable 7, using a stock preparation of 1.0 mg lyophilized ω-ACTXdissolved in 200 μL of methanol (5 mg/mL solution).

TABLE 7 Methanol Treatment Formulations Total Treatment ω-ACTX MethanolVolume 84,375 pmol/g 136.68 μL 5 mg/mL ω-ACTX 113.3 μL   250 μL stock16,875 pmol/g 50 μL 84,375 pmol/g solution 200 μL 250 μL  3,375 pmol/g50 μL 16,875 pmol/g solution 200 μL 250 μL   675 pmol/g 50 μL 3,375pmol/g solution 200 μL 250 μL   135 pmol/g 50 μL 675 pmol/g solution 200μL 250 μL −ve — 250 μL 200 μL

Methanol+DMSO—Solutions of ω-ACTX in methanol were prepared according toTable 8 using a 5 mg/mL stock. The 84,375 pmol/g solution was preparedby diluting the stock mixture into methanol (88.3 μL) before adding DMSO(25 μL) to a final concentration of 10% DMSO. A 10% DMSO in methanolsolution was then prepared and aliquoted as described below for theserial dilution series.

TABLE 8 Methanol/DMSO Treatment Solutions Total Treatment ω-ACTX DMSOMethanol Volume 84,375 pmol/g 136.68 μL 25 μL 88.3 μL  250 μL 5 mg/mLω-ACTX (100%) STOCK in Methanol 16,875 pmol/g 50 μL 84,375 pmol/g — 200μL 250 μL solution (10% DMSO)  3,375 pmol/g 50 μL 16,875 pmol/g — 200 μL250 μL solution (10% DMSO)   675 pmol/g 50 μL 3,375 pmol/g — 200 μL 250μL solution (10% DMSO)   135 pmol/g 50 μL 675 pmol/g — 200 μL 250 μLsolution (10% DMSO) −ve — — 250 μL 250 μL (10% DMSO)

Ethanol+DMSO—Solutions of ω-ACTX in Ethanol were prepared according toTable 9 from a stock preparation of 0.8 mg lyophilized ω-ACTX dissolvedin 160 μL of Ethanol to produce a 5 mg/mL solution. The 84,375 pmol/gsolution was prepared by diluting the stock solution into ethanol (88.3μL) before adding DMSO (25 μL) to a final concentration of 10% DMSO. A10% DMSO/ethanol solution was then prepared and aliquoted as describedbelow for the serial dilution series.

TABLE 9 Ethanol Solution Formulations; order of addition when preparingthe 84,375 pmol/g formulation was ethanol, ω-ACTX stock and finallyDMSO. A 10% DMSO/Ethanol solution was prepared and aliquoted in labeledtubes, and a 5x serial dilution from the 84,375 pmol/g treatment wascarried out. Total Treatment ω-ACTX DMSO Ethanol Volume 84,375 pmol/g136.68 μL 5 mg/mL 25 μL 88.3 μL  250 μL ω-ACTX in Ethanol (100%) 16,875pmol/g 50 μL 84,375 pmol/ — 200 μL 250 μL g solution (10% DMSO)  3,375pmol/g 50 μL 16,875 pmol/ — 200 μL 250 μL g solution (10% DMSO)   675pmol/g 50 μL 3,375 pmol/g — 200 μL 250 μL solution (10% DMSO)   135pmol/g 50 μL 675 pmol/g — 200 μL 250 μL solution (10% DMSO) −ve — — 250μL 250 μL (10% DMSO)

Administration and Application of the Formulation

Each treatment formulation described above was applied to a group oftenCO₂-anesthetized houseflies. Treatment application consisted of a 2 μLdroplet of the respective formulation, pipetted onto the dorsal thoracicbody surface of a fly. Each mixture was vortexed immediately prior toeach application to ensure suspension of precipitate particles.Following treatment, insects were placed in bins with fresh food andwater, allowed to recover, and observed over 60 hours.

Results of methanol or ethanol and DMSO treatments

TABLE 10 Results of treatment with methanol or ethanol and DMSO DeadDead Dead Dead Dead Treatment (6 hrs.) (19.5 hrs.) (24 hrs.) (50 hrs.)(60 hrs.) −ve control Methanol + 0 0 0 0 0 DMSO 135 pmol/g Methanol + 00 0 0 0 DMSO 675 pmol/g Methanol + 0 0 0 0 0 DMSO 3,375 pmol/gMethanol + 0 0 0 0 0 (1 twich) DMSO 16,875 pmol/g Methanol + 0 0 0 (1twch) 0 (1 twch) 0 (1 twch) DMSO 84,375 pmol/g Methanol + 0 3 (3 twch) 3(3 twch) 7 7 (1 twich) DMSO −ve control Methanol 0 0 0 0 0 135 pmol/gMethanol 0 0 0 0 0 675 pmol/g Methanol 0 0 0 0 0 3,375 pmol/g Methanol 00 0 0 0 16,875 pmol/g Methanol 0 0 0 1 (1 twch) 1 84,375 pmol/g Methanol0 0 0 1 1 (5 twch) −ve control Ethanol + 0 0 0 0 0 DMSO 135 pmol/gEthanol + 0 0 0 0 0 DMSO 675 pmol/g Ethanol + 1 1 1 1 1 DMSO 3,375pmol/g Ethanol + 0 0 0 1 0 DMSO 16,875 pmol/g Ethanol + 0 3 (1 twch) 3(2 twch) 7 7 (1 twch) DMSO 84,375 pmol/g Ethanol + 2 7 7 7 7 (1 twch)DMSO Untreated 0 0 0 0 0

Topical treatment of houseflies with a high dose (84,375 pmol/g) ofω-ACTX in ethanol with DMSO was insecticidal causing 30% and 70%mortality at 24 and 50 hours post treatment, respectively. Thetreatments prepared with ethanol were more potent than those preparedwith methanol (70% vs. 30% mortality at 24 hrs. for the highest dose,and 30% vs. 0% mortality in 16,875 pmol/g treatment at 24 hrs.). Theeffect of the highest dose of ω-ACTX in methanol with DMSO was not aspotent as in the previous assay (3 dead, 3 twitching vs. 10 dead at thehighest dose after 24 hrs.).

Methanol/ω-ACTX treatments without DMSO were not insecticidal,suggesting inclusion of an aprotic penetrant or some other type ofmolecular adjuvant is important to the activity of topical preparationsof ω-ACTX.

This experiment shows examples of effective dose ranges of topicallyapplied ω-ACTX diluted in methanol, and what effect the inclusion ofDMSO and ethanol has on the insecticidal activity of the formulation inthe topical bioassay paradigm used.

Example 4 Topical Assay with the Toxin: ω-ACTX-Hv1a

(SEQ ID. NO. 55.) SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCDMolecular weight: 4050. LD₅₀ in House-fly: 90.2 pmol/gFreeze dried aliquots of 1.5 mg toxin prepared from frozen stocks

Topical application: groups often house flies (Musca domestica) fromBenzon research, weighing between 12-20 mg (average mass 16 mg), eachreceived a 2 μL micropipette application of toxin precipitate suspendedin Ethanol-DMSO on the dorsal thoracic surface of the body.

Preparation of stock solutions for topical and per os treatment:

Stock 1 (vortexed preparation): 1 mL ethanol was added to ˜1500 μglyophilized ω-ACTX, and the resulting mixture vortexed vigorously. A 50μL aliquot of the peptide suspension was then removed for topicalapplication assays and kept on ice for ˜2 hrs.; the remainder wasdivided into two ˜475 L aliquots and then kept on ice for ˜2 hrs.

Stock 2 (vortexed and sonicated preparation): 1 mL ethanol was added to˜1500 g lyophilized ω-ACTX, and the resulting mixture vortexedvigorously, and then sonicated ˜10-15 sec., gently ramping up fromintensity setting “0” to setting “5” during this period. A 50 μL aliquotof the peptide suspension was then removed for topical applicationassays and kept on ice for ˜2 hrs.; the remainder was divided into two˜475 μL aliquots and then kept on ice for ˜2 hrs.

Toxin Dose Calculations:

1.5 μg/μL×2 μL/application×10⁶ pg/1 μg×1 pmol/4050 pg×1 fly/0.016g=46,875 pmol/g

Topical application of toxin solutions and results thereof.

Stock solutions of ω-ACTX in Ethanol were prepared as described above.Table 11, below, indicates the recipes used to dilute the stocksolutions for topical application to houseflies:

TABLE 11 Ethanol/DMSO formulations Treatment# 90% EtOH/ Total (dose)ω-ACTX DMSO 10% DMSO Volume 1-vortexed 50 μL ω-ACTX 5 μL — 55 μL (46,875pmol/g) Stock-1 2-vortexed 10 μL treatment 1 — 40 μL 50 μL (9,375pmol/g) solution 3-vortexed + 50 μL ω-ACTX 5 μL — 55 μL sonicatedStock-2 (46,875 pmol/g) 4-vortexed + 10 μL treatment 3 — 40 μL 50 μLsonicated solution (9,375 pmol/g)

Treated flies were kept in containers with ad libitum access to food andwater and mortality (and “twitching” behavior, presumably resulting fromdisruption of physiological norms by action of the toxin) was scoredthereafter as indicated in Table 12 below:

TABLE 12 Results of treament with Ethanol/DMSO Dead Dead Dead Dead DeadDead (40 (48 (76 Treatment N (7.5 hr) (14 hr) (24 hr) hr) hr) hr)  9,000pmol/g 10 0 1 1 2 2 3 vortexed (1 twch) (1 (2 (1 twch) twch) twch)45,000 pmol/g 10 1 6 6 8 8 8 vortexed (2 twitch) (1 twch)  9,000 pmol/g10 2 2 2 3 3 3 sonicated (2 twch) (1 twch) (2 twch) (1 twch) 45,000pmol/g 10 5 8 9 9 9 9 sonicated (2 twch)

This experiment demonstrates that sonicating ω-ACTX suspended in ethanolincreases the topical insecticidal activity of the resulting toxinformulation.

The sonication of ethanol-DMSO precipitates of omega-ACTX-Hv1a enhancesthe insecticidal activity of the omega toxin by increasing the mortalityof contact-treated houseflies, up to 24 hrs. post application.

Example 5 Toxins are

1) ω-ACTX-Hv1a+2:

(SEQ ID. NO. 117) GSSPT CIPSG QPCPY NENCC SQSCT FKENE NGNTV KRCD

has three disulfide bridges: 6-20, 13-24 and 19-38.

Molecular weight: 4199. Injection LD50 in Housefly: 77 pmol/g

rKappa-ACTX-HIv1c:

GSAIC TGADR PCAAC CPCCP GTSCK. AESNG VSYCR KDEP

SEQ ID. NO. 118 has four disulfide bridges: 5-19, 12-24, 15-16, 18-34

Recombinant from pDR2 (pET-32a). Molecular weight: 3912.15

Injection LD50 in Housefly: 389 pmol/g

rU-ACTX-Hv1a+2:

GSQYC VPVDQ PCSLN TQPCC DDATC TQERN ENGHT VYYCR A

SEQ ID. NO. 119 has three disulfide bridges: 5-20, 12-25, 19-39

Molecular Weight: 4570.51

Injection LD₅₀ in Housefly: 81.5 pmol/g

Preparation of mixtures for topical treatment:

Recipes for toxin stocks used to formulate treatment mixtures were asfollows:Stock 1: ω-ACTX-Hv1a+2: an aliquot of 1.5 mg freeze dried toxinsuspended in 850 μL ethanol and sonicated for 10-15 seconds with gentleramp from setting “0” to setting “5” on sonicator to create fineparticles.Stock 2: rU-ACTX-Hv1a+2 an aliquot of 1.5 mg freeze dried toxinsuspended in 900 μL acetone and sonicated for 10-15 seconds with gentleramp from setting “0” to setting “5” on sonicator to create fineparticles.Stock 3: rU-ACTX-Hv1a+2: an aliquot of 1.5 mg freeze dried toxinsuspended in 900 μL methanol and sonicated for 10-15 seconds with gentleramp from setting “0” to setting “5” on sonicator to create fineparticles.Stock 4: rKappa-Hvlc+2: an aliquot of 1.5 mg freeze dried toxinsuspended in 900 μL acetone and sonicated for 10-15 seconds with gentleramp from setting “0” to setting “5” on sonicator to create fineparticles.Stock 5: rKappa-1Hv1c+2: an aliquot of 1.5 mg freeze dried toxinsuspended in 900 μL methanol and sonicated for 10-15 seconds with gentleramp from setting “0” to setting “5” on sonicator to create fineparticles.

Final preparation of mixtures for topical applications was done bymixing stocks with other reagents as listed below:

Control 1—Ethanol+0.05% LI-700-475 μL Ethanol. 25 μL 1% LI-700 inEthanol Control 2—Ethanol+0.01% LI-700-495 μL Ethanol. 5 μL 1% LI-700 inEthanol Control 3—Ethanol+0.1% MSO®-450 μL Ethanol. 50 μL 1% MSO® inEthanol Control 4—Ethanol+0.02% MSO®-490 μL Ethanol. 10 μL 1% MSO® inEthanol

Treatment 1—ω-ACTX-Hv1a+2 Ethanol Precipitate+10% DMSO+0.05% Silwet-425μL

Stock 1 25 μL 1% Silwet in Ethanol. 50 μL DMSO Control 5—Ethanol+10%DMSO+0.05% Silwet—425 μL Ethanol 25 μL 1% Silwet in Ethanol. 50 μL DMSO

Treatment 2—rU-ACTX-Hv1a+2 acetone precipitate+10% DMSO−450 μL Stock 2.50 μL DMSOTreatment 3—rU-ACTX-Hv1a+2 methanol precipitate+10% DMSO−450 μL Stock 3.50 μL DMSOTreatment 4—rKappa-ACTX-Hv1c acetone precipitate+10% DMSO−450 μL Stock4. 50 μL DMSOTreatment 5—rKappa-ACTX-Hvlc methanol precipitate+10% DMSO−450 μL Stock5. 50 μL DMSO

Control 6—Acetone+10% DMSO−450 μL Acetone. 50 μL DMSO

Control 7—Methanol+10% DMSO−450 μL methanol. 50 μL DMSOControl 8—Ethanol+10% DMSO−450 μL Ethanol. 50 μl. DMSO

Administration and application of the formulation.

Topical application of 2 μL, droplets to the ventral abdomen ofhouseflies between 12-18 mg with P10 micropipettes, as described inprevious examples. After application, flies were provided food and waterad libilum and observed for mortality.

Results of the mixtures of topical treatments. Table 13 (below.)

6 hrs. 24 hrs. 42 hrs. Twitch/ Twitch/ Twitch/ Treatment Dead Morib DeadMorib Dead Morib LI-700 (n = 10) Control 1 - Ethanol + 0.05% LI-700 04/0 0 3/1 1 1/1 Control 2 - Ethanol + 0.01% LI-700 0 6/0 1 4/1 1 4/2 MSO(n = 10) Control 3 - Ethanol + 0.1% MSO ® 2 4/1 2 4/0 3 2/1 Control 4 -Ethanol + 0.02% MSO ® 0 7/0 1 5/0 4 1/1 Silwet (n = 10, ~45,000 pmol/g)Treatment 1-ω-ACTX-Hv1a + 2 3/0 7 1/2 10 0/0 2 precipitate + ~10% DMSO +0.05% Silwet Control 5 - Ethanol + ~10% DMSO + 1 0/0 1 0/0 2 0/0 0.05%Silwet Acetone/Methanol Precipitation (n = 10, ~45,000 pmol/g) Treatment2 -rU-ACTX-Hv1a + 2 0 0/0 1 2/0 5 2/0 acetone precipitate + 10% DMSOTreatment 3 - rU-ACTX-Hv1a + 2 0 0/0 2 1/0 5 2/0 methanol precipitate +10% DMSO Treatment 4 - rKappa-ACTX- 0 1/0 0 1/0 5 3/0 Hv1c + 2 acetoneprecipitate* + 10% DMSO Treatment 5 - rKappa-ACTX- 0 1/0 1 1/0 4 0/0Hv1c + 2 methanol precipitate* + 10% DMSO Control 6 - Acetone + 10% DMSO0 0/0 1 0/0 2 0/0 Control 7 - Methanol + 10% DMSO 0 0/0 0 0/0 1 0/0Control 8 - Ethanol + 10% DMSO 0 0/0 0 0/0 0 0/0

Concentrations of LI-700 down to 0.01% resulted in considerabledisruption in the behavior of the treated flies, and possibly somemortality as well. Concentrations of MSO® down to 0.02% resulted inconsiderable disruption in the behavior of the treated flies, andconsiderable (i.e., 30-40%) mortality as well. Silwet, 0.05%, mayslightly potentiate the topical insecticidal activity ofomega-toxin/ethanol/dmso suspensions in this experimental paradigm.Based on results presented here and other undisclosed studiespotentiation would be in the range of 15-20%.

Topically applied formulations of the hybrid and kappa atracotoxin-lsare insecticidal when 90% acetone or 90% methanol is substituted for 90%ethanol. The acetone and methanol formulations of the hybrid toxin maybe slightly less insecticidal than the previously tested ethanolformulation. We believe that acetone and methanol formulations result inequivalent or slightly higher levels of insecticidal activity whencompared to ethanol formulations of kappa toxin.

Example 6 Toxin to Apply Topically is ω-ACTX-Hv1a

(SEQ ID. NO. 60) SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCDhaving a Molecular weight: 4050. LD50 in House-fly: 90.2 pmol/g.Freeze dried aliquots of 1.5 mg toxin prepared from frozen stocks.

Administration and Application of the Formulation

Topical application: groups often house flies (Musca domestica) fromBenzon research, weighing between 12-20 mg (average mass 16 mg), eachreceived a 2 μL micropipette application of toxin precipitate suspendedin Ethanol-DMSO on the dorsal thoracic surface of the body.

Preparation of stock solutions for topical treatment:

Stock 1 (vortexed and sonicated Ethanol preparation): 0.9 mL ethanol wasadded to ˜1500 μg lyophilized ω-ACTX, and the resulting mixture vortexedvigorously, and then sonicated ˜10-15 sec., gently ramping up fromintensity setting “0” to setting “5” during this period. 0.1 mL DMSO wasthen added to the toxin suspension, the resulting mixture was vortexed,and a 100 μL aliquot of the alcohol-DMSO-peptide suspension was thenremoved for topical application assays and kept on ice for ˜0.2 hrs.

Stock 2 (vortexed and sonicated 1-Propanol preparation): 0.9 mL1-propanol was added to ˜1500 hg lyophilized ω-ACTX, and the resultingmixture vortexed vigorously, and then sonicated ˜10-15 sec., gentlyramping up from intensity setting “0” to setting “5” during this period.0.1 mL DMSO was then added to the toxin suspension, the resultingmixture was vortexed, and a 100 μL aliquot of the alcohol-DMSO-peptidesuspension was then removed for topical application assays and kept onice for ˜2 hrs.

Stock 3 (vortexed and sonicated 2-Propanol preparation): 0.9 mL2-propanol was added to ˜1500 μg lyophilized ω-ACTX, and the resultingmixture vortexed vigorously, and then sonicated ˜10-15 sec., gentlyramping up from intensity setting “0” to setting “5” during this period.0.1 mL DMSO was then added to the toxin suspension, the resultingmixture was vortexed, and a 100 μL aliquot of the alcohol-DMSO-peptidesuspension was then removed for topical application assays and kept onice for ˜2 hrs.

Stock 4 (vortexed and sonicated 2-Butanol preparation): 0.9 mL 2-butanolwas added to ˜1500 μg lyophilized ω-ACTX, and the resulting mixturevortexed vigorously, and then sonicated ˜10-15 sec., gently ramping upfrom intensity setting “0” to setting “5” during this period. 0.1 mLDMSO was then added to the toxin suspension, the resulting mixture wasvortexed, and a 100 μL aliquot of the alcohol-DMSO-peptide suspensionwas then removed for topical application assays and kept on ice for ˜2hrs.

Note that each stock was made to a concentration such that a 2 μLapplication of the stock to the body surface of a ˜16 mg housefly wouldresult in a toxin dose of ˜45,000 pmol/g. Hence, in some cases describedbelow, one of the four stocks described above was topically appliedfull-strength to houseflies, but in other cases, five-fold serialdilutions were performed (using the stocks and the corresponding 90%alcohol-10% DMSO solution) in order to obtain a solution that could beused to deliver a lower toxin dose in a 2 μL volume. Negative controlprocedures (indicated as “-ve” in the table below) comprised house fliestreated with 2 μL dorsal throracic applications of solutions of thealcohols in question (diluted to 90% v/v with DMSO).

Topical application of toxin solutions and results thereof:

Stock solutions of co-ACTX in various alcohols were prepared asdescribed above. Table 14 below indicates the stock formulations anddosing used for topical application to houseflies and the observedmortality for the corresponding groups of flies:

TABLE 14 Results of topical application of toxin solutions Whennormalized for mortality observed in negative control groups (dosed withthe corresponding alcohol-DMSO solution), ethanol precipitates of omegatoxin appear to have insecticidal activity as high or higher than anyother toxin-alcohol precipitate tested in this series of experiments. #of Flies Dead Dead Dead Dead Treatment Treated (~16 hr) (24 hr) (52 hr)(64 hr) 1-Propanol −ve 10 0 0 0 0 1-Propanol 10 0 0 1 1 9,000 pmol/g1-Propanol 10 1 2 2 2 45,000 pmol/g (1 twitch) (1 twitch) (1 twitch) (1twitch) 2-Propanol −ve 10 1 2 3 3 (2 twitch) (1 twitch) 2-Propanol 10 34 4 4 9,000 pmol/g (2 twitch) (1 twitch) 2-Propanol 10 5 5 8 8 45,000pmol/g (3 twitch) (3 twitch) 2-Butanol −ve 10 5 7 7 7 2-Butanol 10 6 6 67 9,000 pmol/g (1 twitch) (1 twitch) (2 twitch) Ethanol −ve 7 0 0 0 0Ethanol 10 0 1 1 1 1,800 pmol/g Ethanol 10 2 2 2 2 9,000 pmol/g (2twitch) 1-Octanol −ve 10 10  10  10  10 

90% octanol-10% DMSO, 90% 2-butanol-10% DMSO, and 90% 2-propanol-10%DMSO appear to cause unacceptable levels of background mortality; thelatter could presumably mask mortality due to target site action ofomega toxin in treated houseflies. 90% 1-propanol-10% DMSO does notappear to cause unacceptable levels of background mortality, but it alsodoes not appear to potentiate target site activity of applied toxin aswell as 90% ethanol-10% DMSO.

Example 7 Toxin to Apply Topically

ω-ACTX-Hv1a+2: GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD(SEQ ID. NO. 117). Molecular weight: 4196Freeze dried aliquots of 1.5 mg toxin prepared from frozen stocks

Treatment Mixture Preparation

All treatments were made to 1.5 μg/μL final concentration ofω-ACTX-Hv1a+2. As previously, ethanol was added to ˜1500 μg samples oflyophilized ω-ACTX-Hv1a+2, the resulting mixture was vortexedvigorously, then sonicated ˜10-15 sec., gently ramping up from intensitysetting “0” to setting “5” during this period. Additional ingredientssuch as DMSO, MSO®, water, and Tween 20 detergent were added followingsonication, and the resulting mixtures were vortexed vigorously prior totopical application in order to ensure even mixing of ingredients.Assuming average fly mass of 16 mg (12-20 mg cohort) dosing iscalculated as follows:3 μg/insect×1 μmol/4196 μg×10⁶ pmol/1 μmol×1 insect/0.016 g=44,685pmol/g dose

Recipes for toxin stocks used to formulate treatment mixtures were asfollows:

Stock 1—1.5 mg lyophilized ω-ACTX-Hv1a+2 dissolved in 500 μL ethanol,vortexed and sonicated.Stock 2—1.5 mg lyophilizedo)-ACTX-Hv1a+2 dissolved in 150 μL sterilewater (10 mg/mL).

Topical Assays using ω-ACTX-Hv l a+2 with DMSO:

Recipes for treatment mixtures were as follows:45,000 pmol/g ω-ACTX-Hv1a+2 90% Ethanoll/10% DMSO solution (+-vecontrol)−50 μL Stock 1, 40 μL ethanol, 10 μL DMSO.90% Water/10% DMSO/0.1% Tween 20 (-ve control)−7.5 μL Stock 2, 82.5 μLsterile water, 10 μL DMSO. 1 μL 10% Tween 20.45,000 pmol/g ω-ACTX-Hv1a+2 80% Ethanol/10% Water/10% DMSO−50 μL Stock1, 30 μL ethanol, 10 μL sterile water, 10 μL DMSO.45,000 pmol/g ω-ACTX-Hv1a+2 70% Ethanol/20% Water/10% DMSO−50 μL Stock1, 2 μL ethanol, 20 μL sterile water, 10 μL DMSO.45,000 pmol/g ω-ACTX-Hv1a+2 60% Ethanol/30% Water/10% DMSO−50 μL Stock1, 10 μL ethanol, 30 μL sterile water, 10 μL DMSO.45,000 pmol/g ω-ACTX-Hv1a+2 50% Ethanol/30% Water/10% DMSO−50 μL Stock1, 40 L sterile water, 10 μL DMSO.

For Topical Assay using ω-ACTX-Hv1a+2 with MSO® Surfactant:

5% MSO® (-ve control)−95 μL Ethanol, 5 μL MSO® Concentrate.1.25% MSO® (-ve control)−98.75 μL Ethanol, 1.25 μl. MSO® Concentrate.45,000 pmol/g ω-ACTX-Hv1a+2 5% MSO®−50 μL Stock 1. 45 μL Ethanol, 5 LMSO® Concentrate.45,000 pmol/g ω-ACTX-Hv1a+2 2.5% MSO®−25 μL Stock 1, 23.75 μL Ethanol,1.25 μL MSO® Concentrate.45,000 pmol/g ω-ACTX-Hv1a+2 1.25% MSO®−25 μL, Stock 1, 24.37 μL Ethanol,0.625 μL MSO® ConcentrateAll treatment mixtures were kept on ice for ˜1 hr. prior toadministration and application.

Administration and Application of the Formulation.

2 μL samples of each treatment mixture were spotted onto the dorsalthorax of individual houseflies (10 houseflies treated per mixture). Asecond group often flies were each treated with 2 μL samples of the45,000 pmol/g ω-ACTX-Hv1a+2 in 90% Ethanol/10% DMSO but with thetreatment applied to the ventral abdominal surface of the flies ratherthan the dorsal thoracic surface. After application, flies were kept inplastic containers wells with ad libitum access to food (1:1 mixture ofdry powdered milk and table sugar) and water (presented in soaked cottonballs) and observed every 8-24 hrs. for two days.

Results of Topical application of ω-ACTX-Hv1a+2

Post-application mortality (and “twitching” and “moribund” behavior,both presumably resulting from disruption of physiological norms byaction of the toxin) is summarized in Table 15, below.

TABLE 15 Results of topical assays of DMSO, ethanol and MSO ®preparations of ω-ACTX-Hv1a + 2, administered topically. Dead Dead DeadDead Dead Treatment N (4 hr) (14 hr) (22 hr) (38 hr) (48 hr) -ve90EtOH/10DMSO 10 0 0 0 0 0 45,000 pmol/g 90EtOH/ 10 1 2 3 5 5 10DMSO (1twch) 45,0000 pmol/g 80EtOH/ 10 0 1 2 4 4 10H2O/10DMSO (1 twch) (1 twch)45,0000 pmol/g 70EtOH/ 10 0 0 0 0 0 20H2O/10DMSO 45,0000 pmol/g 60EtOH/10 0 0 0 0 0 30H2O/10DMSO 45,0000 pmol/g 50EtOH/ 10 0 0 0 1 140H2O/10DMSO 45,000 pmol/g 90H2O/ 10 0 1 0 1 1 10DMSO (3 twch) -ve 5%MSO ® 10 0 0 0 0 1 -ve 1.25% MSO ® 10 0 0 1 1 1 (1 morb) (1 morb) 45,000pmol/g 5% MSO ® 10 2 4 4 4 5 (1 twch) (1 morb) (1 morb) (1 twch) 45,000pmol/g 2.5% MSO ® 10 0 3 4 4 5 (1 twch) (1 twch) 45,000 pmol/g 1.25%MSO ® 10 1 1 1 5 6 (1 twch) (1 twch) 45,000 pmol/g 90EtOH/ 10 0 1 5 5 710DMSO TUMMY (3 twch) (1 twch) (1 twch)Table 15 (above). Results of topical assays of DMSO, ethanol and MSO®preparations of ω-ACTX-Hv1a+2, administered topically. Numbers above aredescribed below.

Number 1-addition of 10% water to precipitates of omega toxin inethanol/DMSO solutions appears to reduce topical insecticidal activityof the precipitate under the conditions tested above.

Number 2-addition of 20%, 30%, and 40% water to precipitates of omegatoxin in ethanol/DMSO solutions appears to completely eliminate topicalinsecticidal activity of the precipitate under the conditions testedabove.

Number 3-solvation/dilution of toxin in 90% water/10% DMSO results in asolution with no insecticidal activity under the conditions testedabove.

Number 4-replacement of 10% DMSO with either 1.25% MSO®, 2.5% MSO®, or5% MSO® apparently results in mixtures with significant insecticidalactivity under the conditions tested above.

Number 5-under experimental conditions used above, ventral abdominalapplication of the precipitate (of omega toxin in 90% ethanol/10% DMSO)appears to induce insect mortality with speed and effectiveness similarto, if not greater than, the induction of mortality by dorsal thoracicapplication of the same precipitate mixture. Since ventral abdominalapplication can be executed roughly twice as quickly as dorsal thoracicapplication, this points to a significant technical improvement forfuture topical application bioassays.

Further examples of toxic peptides and the sequence listing.

The toxic insect peptides refers to peptides that are not from toxicinsects, but that are toxic to insects. Their source need not beinsects. In the sequence listing of this application a wide range ofsuitable toxic insect peptides are provided. This small selection ofabout 174 peptides includes representative peptides from spiders,scorpions and plants. Sequences 1-140 are from funnel web spiders,sequences 141 to 171 are from scopions and sequences 172 to 174 are fromplants. The sequence listing includes peptide examples where the sourceis Oldenlandia affinis which is known to produce a cyclic peptidereferred to at a “Kalata” type peptide. The Oldenlandia plant family isknown to produce peptides having insecticidal activity. Otherinsecticidal peptides from plants are known. Numerous venomus spiderpeptides, which are discussed throughout this application are alsoprovided.

1. A process for increasing the topical insecticidal activity of a toxicpeptide comprising: a) preparing a topical toxic peptide; b) mixing afirst solvent with said peptide, wherein said first type of solvent iseither a polar aprotic solvent or a polar organic solvent; c) mixing asecond type of solvent with said first solvent peptide mixture, whereinsaid second type of solvent is not the same type of solvent as saidfirst type of solvent and is either a polar aprotic solvent or a polarorganic solvent.
 2. The process of claim 1 wherein said polar aproticsolvent is selected from: dimethyl sulfoxide (DMSO), dimethylformamide,dioxane and hexamethylphosphorotriamide.
 3. The process of claim 2wherein said polar organic solvent is selected from acetone, methanol,ethanol, propanol and all its isomers, methyl ethyl ketone, diethylketone, acetonitrile, and ethyl acetoacetate.
 4. The process of claim1-3 wherein said polar aprotic solvent is selected from: dimethylsulfoxide (DMSO) or MSO® and said polar organic solvent is selected fromacetone, methanol, ethanol and propanol.
 5. The process of claim 1-3wherein said peptide is lyophylized before the first solvent is mixedwith the peptide.
 6. The process of claim 1-3 wherein said first solventis a polar aprotic solvent.
 7. The process of claim 1-3 wherein saidpolar aprotic solvent is from about 60% to about 99% percent, and saidpolar organic solvent is about 40% to about 1%, of the total solventvolume.
 8. The process of claim 1-3 wherein said polar aprotic solventis from about 75% to about 95%, and the polar organic solvent is about25 to about 5%, of the total solvent volume.
 9. The process of claim 1-3wherein said polar aprotic solvent is from about 80% to about 90% of thetotal solvent volume and the polar organic solvent is from about 20% toabout 10% of the total solvent volume.
 10. The process of claim 1-3wherein said peptide is any topical toxic peptide selected from aselected or derived from a toxic peptide from any species of spider,scorpion, snake, mite, snail, slug or plant and any peptides having 50%or greater homology to any such peptide.
 11. The process of claim 1-3wherein the length of said peptide is from 10 to 200, amino acids inlength.
 12. The process of claim 1 wherein said peptide has from 1-5,internal disulfide bonds
 13. The process of claim 1 wherein said peptideis selected from a spider or scorpion.
 14. The process of claim 1wherein said peptide is selected from the Australian funnel web spiderof genus Atrax or Hadronyche.
 15. The process of claim 1 wherein saidpeptide is selected from any sequence in the sequence listing or anysequence having 50% or greater homology to any of the listed sequences.16. The process of claim 15 wherein said peptide is selected from any ofthe sequences in the sequence listing.
 17. The process of claim 16wherein said peptide is selected from any of the following sequences:SEQ ID NO: 60, SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO:
 119. 18. Apeptide formulation comprising: a) a peptide; b) a polar organicsolvent; c) a polar aprotic solvent or adjuvant; d) wherein said polarorganic solvent comprises from about 70 to about 99 percent (%) of thefinal volume of the formulation; e) wherein said polar aprotic solventor adjuvant comprises from about 30, to about 1 percent (%) of the finalvolume of the suspension; f) an optional water phase, wherein said waterphase comprises from 0 (zero), to about 10 percent (% o) of the finalvolume of the suspension; and
 19. A peptide described in claim 10, madespecial by the process described in claim
 1. 20. The control of aninsect with the special toxic peptide formulation of claim 18 whereinthe special toxic peptides and formulations are applied to the insect'senvironment.